Ubiquitin (Ub) is 76-residue protein that exists in cells either free or conjugated to many other proteins. Regulated degradation of intracellular proteins by the Ub-proteasome system plays a central role in an astounding multitude of biological processes, including cell growth and differentiation, signal transduction, and responses to stress. One universally present Ub/proteasome-dependent proteolytic system is the N-end rule pathway. The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Studies supported by this grant (GM31530), currently in its 22rid year of funding, have yielded several important insights into the mechanisms and functions of the N-end rule pathway, as described in the Progress Report. Over the last 4 years, this grant supported our studies of the mouse N-end rule pathway. The objective of research described in the present renewal is to continue these studies, and to further advance the understanding of the N-end rule pathway and related aspects of the mammalian Ub system. SPECIFIC AIMS: 1) Construction and functional analyses of mouse strains (and cells derived from them) in which the expression of the ATE1 encoded Arg-tRNA-protein transferases (R transferases) is selectively and conditionally abolished (or induced) in specific cell lineages during embryogenesis, or postnatally. 2) Analysis of chromosome stability and regulation of apoptosis in mouse ATE1(-/-) cells. 3) Analysis of chromosome stability and regulation of apoptosis in mouse NTAN 1(-/-), UBRI(-/-), UBR2(-/-), UBR3(-/-), and double-mutant [UBRI(-/-)UBR2(-/-)] cells. 4) Identification of ATEl-dependent circuits (i.e., the circuits that involve N terminal arginylation) through the identification of mouse genes whose expression is significantly altered during embryonic development in ATE1(-/-) embryos, specifically at the time of a strong spike of ATE1 expression in +/+ embryos that peaks on day E8.5 5) Further identification of physiological substrates of the mammalian N-end rule pathway. The projects of this Aim include a tag-based coprecipitation-mass spectrometry approach to identifying specific ligands of the mouse UBR1, UBR2 and UBR3 Ub ligases (E3 proteins).